The overall objective of this proposal is to evaluate the anticoagulant properties of a family of novel tick salivary proteins Ixodes scapularis, the black-legged tick, circumvents host hemostatic mechanisms as it engorges on vertebrate hosts. This tick's saliva contains potent anticoagulants, including a Factor Xa inhibitor. Purified salivary proteins with anticoagulant activity were subjected to N-terminal sequencing. A corresponding tick salivary cDNA was cloned that encoded a 96 kDa protein, designated Salp9A. This protein is homologous to Salp14, which we previously identified by immunoscreening a tick salivary gland cDNA expression library. Additional protein and cDNA sequencing indicate that I scapularis salivary glands express at least ten different Salp14-related proteins. We expressed and purified recombinant Salp14, and found that it inhibits coagulation and Factor Xa. These data suggest that we have discovered a new family of anticoagulants. In Phase I experiments we will obtain full-length cDNAs encoding selected Salp14 family members. Recombinant proteins will be expressed from these cDNAs. The recombinant proteins will be compared to partially purified salivary anticoagulants in anticoagulant and protease inhibition assays. These studies will relate structure and function, and provide a rational basis for selecting anticoagulant molecules for Phase II animal studies. [unreadable] [unreadable]